Insulin resistance is a marker for increased risk of Non-Insulin- Dependent Diabetes (NIDDM), Gestational Diabetes (GDM), hypertension, and cardiovascular disease, and is a major cause of glucose intolerance in NIDDM and GDM. However, the degree to which insulin sensitivity is determined by genetic and environmental factors is unknown, and molecular mechanisms causing insulin resistance have not been fully elucidated. In the previous cycle, the P1 found that insulin resistance in NIDDM was due to depletion of GLUT 4 glucose transporters (the major insulin- responsive isoform) in adipocytes, while GLUT 4 levels were normal in skeletal muscle indicating that either GLUT 4 translocation (to the cell surface) or functional activity was impaired. The overall goal in the proposed studies is to define the heritability of insulin resistance and to identify mechanisms which reduce glucose transport system activity in humans with and without insulin-resistant disease processes. Heritability of insulin resistance will be assessed by measuring maximally-stimulated glucose uptake in monozygotic and dizygotic twins, and estimating genetic variance based on intraclass differences and model-fitting path analyses. Muscle fiber composition and GLUT 4 content, resting metabolic rate, and body composition will also be measured so that interrelationships can be assessed within the context of the MZ/DZ twin model. Other studies will characterize molecular mechanisms causing insulin resistance in NIDDM and GDM. In NIDDM, adipocytes will be studied to determine whether nuclear proteins suppress GLUT 4 gene transcription, and the responsible cis and trans elements "will be identified. Studies in skeletal muscle will demonstrate whether insulin resistance in NIDDM and GDM is due to impaired translocation or reduced activity of GLUT 4. GLUT 4 recruitment will be measured by exploiting our ability to biopsy muscle fibers held at fixed length and to photolabel (3H-ATB-BMPA) cell-surface GLUT 4. GLUT 4 activity will be assessed in native membrane vesicles and after reconstitution in synthetic liposomes. Preliminary data in GDM indicate that GLUT 4 accumulates in an alternative membrane compartment where it then becomes refractory to insulin-mediated translocation in adipocytes. This defect will be further characterized in both fat (GDM) and muscle (NIDDM and GDM) by biochemically analyzing immuno-purified GLUT 4 vesicles including their content of small molecular weight G-proteins (smg's), assessing the subcellular distribution of other insulin-regulated proteins (GLUT 1, IGF 11 receptors), and morphologically localizing GLUT 4 using immuno- gold/EM. Finally, insulin-mediated recruitment and activity of GLUT 4 will be assessed in muscle biopsies from two groups of MZ twins concordant for upper and lower quartiles of insulin sensitivity to determine if mechanisms causing insulin resistance in subjects without overt disease are similar to those operative in diabetes. Major hypotheses are supported by preliminary data and include: 1) Insulin resistance is a highly inherited trait due in part to genetic factors which determine skeletal muscle fiber composition and GLUT 4 content; 2) In NIDDM, specific transcription factors in adipocytes repress GLUT 4 gene transcription; 3) In NIDDM and GDM, insulin resistance in skeletal muscle is due to impaired insulin-mediated translocation of GLUT 4 to the cell surface; 4) the translocation defect is associated with abnormal subcellular localization of GLUT 4 vesicles and smg's. Thus, these studies will combine basic and clinical research methodologies to directly study heritability and mechanisms of human insulin resistance, and will further elucidate the pathogenesis and role of insulin resistance in human diseases.